Diagnostic ultrasonic imaging is based on the principle that waves of sound energy can be focused upon an area of interest and reflected in such a way as to produce an image thereof. The ultrasonic transducer is placed on a body surface overlying the area to be imaged, and ultrasonic energy in the form of sound waves is directed toward that area. As ultrasonic energy travels through the body, the velocity of the energy and acoustic properties of the body tissue and substances encountered by the energy determine the degree of absorption, scattering, transmission and reflection of the ultrasonic energy. The transducer then detects the amount and characteristics of the reflected ultrasonic energy and translates the data into images.
As ultrasound waves move through one substance to another there is some degree of reflection at the interface. The degree of reflection is related to the acoustic properties of the substances defining the interface. If these acoustic properties differ, such as with liquid-solid, liquid--liquid or liquid-gas interfaces, the degree of reflection is enhanced. For this reason, gas-containing contrast agents are particularly efficient at reflecting ultrasound waves. Thus, such contrast agents intensify the degree of reflectivity of substances encountered and enhance the definition of ultrasonic images.
Ophir and Parker describe two types of gas-containing imaging agents: (1) free gas bubbles; and (2) encapsulated gas bubbles (Ultrasound in Medicine and Biology 15(4):319-333 (1989)), the latter having been developed in an attempt to overcome instability and toxicity problems encountered using the former. Encapsulated gas bubbles, hereinafter referred to as "microspheres," are composed of a microbubble of gas surrounded by a shell of protein or other biocompatible material. One such imaging agent is ALBUNEX.RTM. (Molecular Biosystems, Inc., San Diego, Calif.) which consists of a suspension of air-filled albumin microspheres.
Generally, microspheres of a particular gas exhibit improved in vivo stability when compared to free bubbles of the same gas. However, most microspheres still have relatively short in vivo half lives which compromise their usefulness as contrast agents. This instability in vivo was thought to result from the collapse or breakdown of the shells under pressure resulting in rapid diffusion of the gas from the microspheres. Thus, many recent efforts have centered on improvements to the shell as a way of increasing in vivo stability. Known improvements relating to protein-shelled microspheres include coating the protein shell with surfactants (Giddy, WO 92/05806) and chemical cross-linking of the protein shell (Holmes et al., WO 92/17213).
Additional efforts directed toward improving microsphere stability include the use of non-proteinaceous shell-forming materials. Bichon et al. (European Patent Application 458,745A1) and Schneider et al. (Inv. Radiol. 27:134-139 (1992)) describe the production of polymeric "microballoons" made of interfacially deposited polymers encapsulating various gases such as carbon dioxide, nitrous oxide, methane, freon, helium and other rare gases. Klaveness (WO 92/17212) describes the use of chemically-linked, non-proteinaceous amphiphilic moieties encapsulating "air, nitrogen, oxygen, hydrogen, nitrous oxide, carbon dioxide, helium, argon, sulfur hexafluoride and low molecular weight, optionally fluorinated, hydrocarbons such as methane, acetylene or carbon tetrafluoride." Erbel et al. (U.S. Pat. No. 5,190,982) describe the use of polyamino-dicarboxylic acid-co-imide derivatives.
More recently, Schneider et al. (U.S. Pat. No. 5,413,774) have demonstrated that microspheres containing gases with certain physical properties have improved stability. It is theorized that microsphere instability is caused by the increase in pressure to which microspheres are exposed once they are introduced into the circulatory system. Although Schneider et al. do not speculate as to the mechanism responsible for their observation of enhanced pressure resistance, we believe it is due to the effects of gas solubility on the rate of gas exchange with the aqueous environment.
According to Henry's law, the solubility of a given gas in solution increases as pressure increases. When a bubble of gas in solution is subjected to pressure, the rate of gas exchange between the gas in the bubble and the surrounding solution will increase in proportion to the amount of pressure, and the bubble of gas will eventually become completely solubilized. The more insoluble the gas is in the surrounding solution, the longer it will take for a bubble to become completely solubilized.
If the bubble of gas is surrounded by a shell, i.e., in the form of a microsphere, the effects of gas exchange are still observed, since microsphere shells do not completely eliminate contact between gas in the microsphere and the surrounding solution. Hence, when microspheres suspended in solution are subjected to pressure, the gas inside the microspheres eventually becomes solubilized in the surrounding solution which results in collapse of the microspheres.
In order to inhibit the exchange of gas in the microsphere center with the surrounding aqueous environment, the present invention describes a process for increasing the hydrophobicity of the microsphere shell by forming a solid or liquid hydrophobic barrier on the inner surface of the microsphere shell. Microspheres formed by this process will exhibit decreased water permeability and thus enhanced resistance to pressure instability due to gas exchange.
Disclosure of the Invention
The present invention provides insoluble gas-filled microspheres containing a liquid or solid hydrophobic barrier on the inner surface of the microsphere shell which increases the overall hydrophobicity of the microsphere. In particular, the present invention provides for a process of producing microspheres comprising a shell formed from amphiphilic, biocompatible material surrounding a microbubble of at least one biocompatible insoluble gas and a liquid or solid hydrophobic compound barrier formed at the inner surface of the microsphere shell, said barrier decreasing the permeability of the shell and the rate of gas exchange with the aqueous environment surrounding the microsphere. In the present invention, a biocompatible insoluble gas suitable for use with the present invention is saturated with a hydrophobic "sealer" compound with a boiling point above room temperature. Upon cooling to room temperature, the hydrophobic compound will condense on the hydrophobic inner shell surface of the microsphere thereby creating an additional hydrophobic barrier and increasing the overall hydrophobicity of the microsphere shell. The hydrophobic compound can be inert and includes, but is not limited to, members of the hydrocarbon, halogenated hydrocarbon or perfluorocarbon series. The hydrophobic compound can have a linear, branched or cyclic molecular structure.
Hydrophobic compounds within the present invention, when introduced by a water-insoluble gas, can be reactive and capable of covalently bonding to reactive amino acid side chains in the protein of the shell. If some of the reactive material undergoes hydrolysis before it has an opportunity to covalently bond with the proteinaceous shell, the resulting hydrolysis product would also form a hydrophobic deposit on the inner surface of the microsphere and act as a sealer.
Alternatively, a polymerizable, low-boiling monomer can be introduced with a water-insoluble gas. Hydrophobic monomers within the present invention polymerize inside the microsphere, optionally as a result of reaction with a chemical initiator or light, to form a polymer layer on the inner surface of the microsphere. The polymeric material formed includes, but is not limited to substituted polyethylenes, polystyrene and the like. Preferred polymeric materials include polyacrylate and polymethacrylate.
In the cases described above, a hydrophobic layer is formed which is chemically and/or physically attached to the inner surface of the proteinaceous shell. In another aspect of the invention, the hydrophobic compound forms a sponge-like structure within the proteinaceous shell occupying some or all of the space within the microsphere and contains biocompatible insoluble gases within the interstices.
The amount of hydrophobic compound introduced in the shell is an amount sufficient to decrease the permeability of the microsphere shell to the aqueous environment. The decrease in permeability of the shell results in slower rate of gas exchange with the aqueous environment and is evidenced by enhanced pressure resistance of the microsphere. The amount of hydrophobic compound introduced into the microsphere by the insoluble gas can be controlled by its partial vapor pressure. Raising or lowering the temperature of a bath containing the liquid hydrophobic compound will increase or decrease, respectively, the size of the hydrophobic layer. The partial vapor pressure is dependent on the temperature of the bath containing the hydrophobic compound. For example, insoluble gas can be saturated with perfluorodecalin (bp. 142.degree. C. at 760 mm Hg) by bubbling the gas through perfluorodecalin that is maintained at 75.degree. C. by standing in a temperature bath. The partial vapor pressure attained will be approximately 100 mm Hg. The saturated gas is then mixed with 1% human serum albumin to form microspheres by a cavitation process.
Microspheres formed by ultrasonication generally have a mean diameter of 3-5 microns and a shell thickness of approximately 20 nm. While the specific amount of hydrophobic compound necessary to decrease the permeability of the microsphere will vary with the hydrophobic compound and the gas, the gas generally should be saturated with the hydrophobic compound to give a partial pressure in the range of about 10 to about 650 mm Hg, preferably about 50 to about 250 mm Hg.
It is anticipated that with increasing boiling point, the sealer compounds become more efficient and therefore smaller amounts would be required to obtain the same stabilizing effect. The reason for this is that higher boiling compounds are adsorbed more strongly to the naturally hydrophobic inner surface of the microsphere.
Gases suitable for use within the present invention are pharmacologically acceptable and insoluble and include, but are not limited to, sulfur hexafluoride, perfluoromethane, perfluoroethane, perfluoropropane and perfluorobutane and the like.
Suitable microsphere shell material includes proteins and synthetic amino acid polymers. The material is preferably a protein, and more preferably human serum albumin.
The microspheres of the present invention may be made by known methods used to make conventional gas-filled microspheres such as sonication, mechanical cavitation using a milling apparatus, or emulsion techniques.
Modes of Carrying Out the Invention
Microspheres which contain insoluble gases are known to be more pressure resistant than equivalent microspheres containing soluble gases. This is because the rate of exchange of the gas with the surrounding aqueous environment is slower for an insoluble gas than a soluble gas. However, even insoluble gases eventually escape into the aqueous environment which diminishes the shelf life of insoluble gas-containing microspheres.
The present invention relates to the discovery that the stability of insoluble gas-containing microspheres can be improved by rendering the microsphere shells more hydrophobic to diminish the ability of the aqueous environment to come in contact with the microsphere's gas core. This is accomplished by using a process of making microspheres which increases the hydrophobicity of microsphere shells by creating a liquid or solid hydrophobic barrier on the inner surface of a microsphere shell. Such a barrier is produced by saturating an insoluble gas suitable for use within the present invention with a hydrophobic compound, such as a hydrocarbon or perfluorocarbon or the like, at an elevated temperature. Microspheres are then prepared using conventional cavitation techniques and the hydrophobic compound saturated in the gas will condense and/or react with the shell material and form a liquid or solid hydrophobic barrier on the inner surface of the microsphere shell.
Suitable hydrophobic compounds within the present invention include, but are not limited to, hydrocarbons of the general formula, C.sub.n H.sub.2n+2, wherein n=6-12, such as octane (bp=126.degree. C.) or isooctane (bp=99.degree. C.), linear or branched perfluorocarbons of the general formula C.sub.n F.sub.2n+2, wherein n=5-12, such as perfluoropentane (bp=29.degree. C.), perfluorohexane (bp=60.degree. C.), perfluoroheptane (bp=80.degree. C.), perfluorooctane (bp=100.degree. C.) or 1-bromoperfluorooctane (bp=142.degree. C.) and cyclic perfluorocarbons such as perfluoromethylcyclohexane (bp=76.degree. C.), perfluorodecalin (bp=142.degree. C.), and octafluorotoluene (bp 105.degree. C.). Inert hydrophobic compounds within the present invention include, but are not limited to, perfluorinated alcohols such as 1H,1H-heptafluoro-1-butanol (bp=96.degree. C.), 1H,1H,7H-dodecafluoro-1-heptanol (bp=170.degree. C.), ethers such as 2,3,4,5,6-pentafluoroanisole and esters such as alkyl perfluoroalkanoates.
Reactive hydrophobic compounds include, but are not limited to, active esters such as linear alkyl trifluoracetates or acyl chlorides with the general formula C.sub.n H.sub.2n+1 COCl, wherein n=4-10, such as hexanoyl chloride (bp=152.degree. C.) or C.sub.n F.sub.2n+1 COCl, wherein n=2-10, such as perfluorooctanoyl chloride (bp=132.degree. C.) and cyclic compounds such as pentafluorobenzoyl chloride (bp=159.degree. C.). Acid chlorides of some perfluoro alkanedioic acids, such as tetrafluorosuccinyl chloride, hexafluoroglutaryl chloride and octafluoroadipoyl chloride, which are bifunctional and capable of crosslinking proteinaceous material can also serve as hydrophobic compounds. Polymerizable hydrophobic compounds within the present invention include, but are not limited to, styrenes such as pentafluorostyrene (bp=140.degree. C.), alkyl acrylate, CH.sub.2 =CHCOOR, wherein R=C.sub.n H.sub.2n+1 or C.sub.n F.sub.2n+1, wherein n=1-6 and the corresponding alkyl methacrylates and fluoroalkyl methacrylates. Also within the present invention are the alkyl-2-cyanoacrylates.
Suitable shell material must be amphiphilic, i.e., containing both hydrophobic and hydrophilic moieties. It must also be capable of forming a thin layer or skin around the encapsulated gas, which will generally result in hydrophilic groups oriented externally and hydrophobic groups oriented internally. When microspheres are produced to contain insoluble gas, this orientation is believed to be enhanced by the presence of the insoluble gas during microsphere formation.
Protein shells may also optionally incorporate proteins, amino acid polymers, carbohydrates, lipids, sterols or other substances useful for altering the rigidity, elasticity, biodegradability and/or biodistribution characteristics of the shell. The rigidity of the shell can also be enhanced by cross-linking, for example, with irradiation.
Protein shell material includes both naturally-occurring proteins and synthetic amino acid polymers which herein are both generally referred to as being in the class of shell materials described as "proteins". Examples of naturally-occurring proteins include gamma-globulin (human), apo-transferrin (human), beta-lactoglobulin, urease, lysozyme, and serum albumin. Synthetic amino acid polymers can optionally be in the form of block or random co-polymers combining both hydrophobic and hydrophilic amino acids in the same or different chains. Moreover, amino acid polymers can optionally be fluorinated.
The present invention also contemplates the attachment of target-specific moieties to the outer shell material of microspheres. Microspheres within the present invention provide a superior delivery vehicle for such target-specific moieties due to the increased stability of the microspheres. Such increased in vivo stability insures delivery of target-specific moieties to targeted organs or cells via lengthy routes of administration.
The introduction of the hydrophobic compound in the shell is accomplished by forming microspheres at elevated temperatures in the presence of an insoluble gas saturated with a hydrophobic compound having a boiling point above 20.degree. C. For example, perfluoropropane can be saturated with perfluoropentane (bp=30.degree. C.), perfluorohexane (bp=60.degree. C.), perfluoroheptane (bp=80.degree. C.), perfluorooctane (bp=100.degree. C.) or perfluorodecalin (bp=142.degree. C.). The saturated gas is maintained at a temperature above the bath temperature and mixed with 1-5% human serum albumin to form microspheres by a cavitation technique.
Microspheres containing the hydrocarbon barrier may be made by known methods used to make conventional gas-filled microspheres such as sonication, mechanical cavitation using a milling apparatus, or emulsion techniques. Such techniques are exemplified in U.S. Pat. Nos. 4,957,656; 5,137,928; 5,190,982; 5,149,543: PCT Application Nos. WO 92/17212; WO 92/18164; WO 91/09629; WO 89/06978; WO 92/17213; GB 91/00247; and WO 93/02712: and EPA Nos. 458,745 and 534,213 which are incorporated herein by reference.
Gases suitable for use within the present invention are pharmacologically acceptable, i.e., biocompatible and minimally toxic to humans and insoluble. The term "insoluble gas" as used herein intends gases and mixtures of gases which are entirely insoluble, as well as mixtures of gases which contain minor amounts (less than 20% v/v) of soluble gas(es) such as air. Insoluble gases are necessary to achieve a desired slow rate of gas exchange with the aqueous environment. The term "biocompatible" means the ability of the gas to be exhaled or metabolized without the formation of toxic by-products. The term "gas" refers to any compound which is a gas or capable of forming gas at the temperature at which imaging is being performed (typically normal physiological temperature) or upon application of ultrasound energy. The gas may be composed of a single compound or a mixture of compounds. The gas is preferably a perfluorocarbon which is insoluble in water, which intends a solubility of less than 0.01 mL of gas per mL of water at atmospheric pressure and a temperature of 25.degree. C. Examples of perfluorocarbon gases suitable for use within the present invention are perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane and perfluoroisobutane. This degree of insolubility results in maximum stability in vitro and persistence in vivo. Solubility can be determined by any appropriate method. See, for example, Wen-Yang Wen et al. (1979) J Solubility Chem. 8(3):225-246.
Microspheres made by processes within the present invention are echogenic (i.e., capable of reflecting sound waves) being composed of material having acoustic properties which are significantly different from those of blood or tissue. The maximum size (mean diameter) of the microsphere is defined by that size which will pass through the pulmonary capillaries. In the case of humans, that size will typically be less than about 10 micrometers. Correspondingly, the minimum size is that which will provide efficient acoustic scattering at the ultrasonic frequencies typically used for ultrasonic imaging. (The frequency may vary with the mode of imaging, e.g., transthoracic, transesophageal, and will normally be in the range of 2-12 MHz.) The minimum size will typically be about 0.1 micrometers. The typical mean size of the microspheres used in the invention method will be about 2 to about 7 micrometers. This size will permit their passage through capillaries, if necessary, without being filtered out prior to reaching the area to be imaged (e.g., where a peripheral venous injection site is used). Thus, microspheres within the present invention will be capable of perfusing tissue and producing an enhanced image of the tissue, organs and any differentiation between well-perfused and poorly-perfused tissue, without being injected into the arteries or directly into the area to be imaged. Accordingly, they may be injected into a peripheral vein or other predetermined area of the body, resulting in considerably less invasion than the arterial injections required for an angiogram.
The microsphere suspensions may be stored in sterile glass vials after manufacturing, or they may be stored in syringes, which are ready for use in administering the microsphere suspensions.
Microspheres made by processes within the present invention may be used for imaging a wide variety of areas. These areas include, but are not limited to, myocardial tissue, liver, spleen, kidney, and other tissues and organs presently imaged by ultrasonic techniques. Use of microspheres within the present invention may result in an enhancement of such currently obtainable images.
In terms of method of operation, the use of the subject microspheres would be the same as that of conventional ultrasonic contrast agents. The amount of microspheres used would be dependent on a number of factors including the choice of liquid carriers (water, sugar solution, etc.), degree of opacity desired, areas of the body to be imaged, site of injection and number of injections. In all instances, however, sufficient microspheres would be used in the liquid carrier to achieve enhancement of discernible images by the use of ultrasonic scanning.
For use in conventional or harmonic ultrasound imaging, the suspension of microspheres is injected into a peripheral vein, either as a bolus or continuously infused over a period of time, such as one to ten minutes, at about 0.05 to 0.5 cc per kg body weight. Ultrasonic energy is applied either continuously or intermittently (i.e., pulsed) to the tissue/organ to be imaged, and reflected energy is collected and translated into an image using conventional, commercially available ultrasound imaging equipment.
Two dimensional (2-D) or multidimensional (e.g. three-dimensional (3-D)) echocardiography equipment and procedures may be used to acquire the image. Such procedures and equipment are conventional. Three techniques used to acquire 3-D images are as follows: In the first, a standard transducer is used to collect tomographic images. The transducer is mounted on a track and collects images as it moves along the track. The speed of motion along the track is defined, so that the spacing between tomographic images is known. The collection of slices are then melded together to obtain a 3-D image. In the second, a standard transducer is also used to collect tomographic images. Attached to the transducer is a sensor that is able to report the spatial position of the transducer, so that the relative orientation of various images are known and the images can be melded together to generate a 3-D image. In the third, the transducer consists of a two dimensional array of elements. A one dimensional array of elements is able to acquire a tomographic image; the added dimension allows scanning in the third dimension.
The invention is further illustrated by the following examples. These examples are not intended to limit the invention in any manner. The disclosures of the publications, patents, patent applications, and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure.